CN109468258A - The tolerance propionate bacteria acclimation method in one plant of cud source - Google Patents

The tolerance propionate bacteria acclimation method in one plant of cud source Download PDF

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CN109468258A
CN109468258A CN201811360010.0A CN201811360010A CN109468258A CN 109468258 A CN109468258 A CN 109468258A CN 201811360010 A CN201811360010 A CN 201811360010A CN 109468258 A CN109468258 A CN 109468258A
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propionate
tolerance
cud
culture
plant
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葛蔚
柴超
张勇
王�忠
唐超
张文丽
王清吉
林英庭
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Qingdao Agricultural University
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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Abstract

The invention discloses the tolerance propionate bacteria acclimation methods in one plant of cud source, take rumen fluid, sodium propionate and minimal medium is added in constant-temperature table, it is protected from light culture, gradually change propionic acid na concn, it is gradually inoculated with, completes to cultivate under the conditions of the sodium propionate of various concentration, bacterium solution obtained is crossed isolated object bacteria on solid medium.The beneficial effects of the invention are as follows obtained the tolerance propionate bacteria bacterial strain in one plant of cud source by screening domestication, the bacterial strain, which has, is applied to feed fermentation, the mainly aerobic fermentation of herbage and stalk plays the role of improving feed nutritive value and some of the contaminants of degrading.

Description

The tolerance propionate bacteria acclimation method in one plant of cud source
Technical field
The invention belongs to technical field of microbial fermentation, are related to a kind of screening, domestication and application for being resistant to propionate bacterial strain, Combination agent propionate is applied to feed by the bacterial strain Gordonia paraffinivorans ZM052 for screening and taming Fermentation, the mainly aerobic fermentation of herbage and stalk are played the role of improving feed nutritive value and some of the contaminants of degrading.
Background technique
In the field of microbe application, people are primarily upon the scope of application of bacteriostatic agent to the use of bacteriostatic agent, and are directed to It goes the report for obtaining corresponding tolerance bacterium less in a kind of bacteriostatic agent, is had not seen from the point of view of current document using propionate and screened With the relevant report of the bacterium of domestication tolerance propionate, not seen in the report for systematically discussing bacteriostatic agent with it and being resistant to bacterium use in conjunction Road.The method that the technical program is intended to establish the bacteria screening and domestication of tolerance propionate is resistant to propionic acid for system development in the future Salt microorganism, joint propionate and tolerance propionate microorganism carry out using offer technical support.
Summary of the invention
The purpose of the present invention is to provide the tolerance propionate bacteria acclimation methods in one plant of cud source, solve existing skill Without bacteriostatic agent and the problem of tolerant microorganisms use in conjunction present in art.
The beneficial effects of the invention are as follows obtained the tolerance propionate bacteria in one plant of cud source by screening domestication GordoniaparaffinivoransZM052, it was confirmed that the bacterial strain has good growth performance in propionate；It constructs resistance to Screening and acclimation method by propionate bacteria, and propose the side for propionate being used in combination and resistance to propionate microorganism is applied Method；Observe that the bacterial strain has life in sodium carboxymethylcellulose, alkaline lignin, naphthalene, phenanthrene, anthracene, the culture medium that pyrene is carbon source It is long, thus indicate that the bacterial strain has applied to feed fermentation, the mainly aerobic fermentation of herbage and stalk, playing improves feed battalion Support the effect of value and degradation some of the contaminants.
The technical scheme adopted by the invention is that: it takes rumen fluid, sodium propionate and minimal medium is added in constant-temperature table, It is protected from light culture, propionic acid na concn is gradually changed, is gradually inoculated with, completes to cultivate under the conditions of the sodium propionate of various concentration, will be obtained Bacterium solution cross on solid medium isolated object bacteria.
Further, it takes the rumen fluid of 5ml in 100ml triangular flask, the sodium propionate of 0.1-1% and the inorganic salts of 40ml is added Culture is based on constant-temperature table 120-150rpm, is protected from light culture at 30-39 DEG C, and growth situation is observed after 2-5 days；It gradually changes Propionic acid na concn, is gradually inoculated with, and completes to cultivate 1 week under the conditions of the sodium propionate of various concentration as 1 round, completes 3-5 round Culture；Bacterium solution obtained is crossed isolated object bacteria on solid medium.
Further, propionic acid na concn is 0.25%, 0.5%, 0.75%, 1.0%, carries out the culture of 4 rounds, cultivates item Part is constant-temperature table 150rpm, is protected from light culture at 35 DEG C.
Further, the various salinity of minimal medium are as follows: MnSO40.02-0.05mg/L、ZnSO40.02-0.05mg/L、 (NH4)2MoO40.02-0.05mg/L、FeCl30.1-0.4mg/L、CaCl220-50mg/L、MgSO450-80mg/L、K2HPO490- 120mg/L、Na2HPO4100-160mg/L、KH2PO430-50mg/L、NH4Cl15-50mg/L。
Further, object bacteria is taken early period to be inoculated with shaking flask culture in the sodium propionate minimal medium containing 0.1-1%, gradually Propionic acid na concn is improved to 1%；Later period uses the sodium propionate minimal medium shaking flask culture of 1-5%, wherein according to glucose: Glucose is added in the ratio that sodium propionate is 1:20-1:5, and each propionic acid na concn cultivation cycle is 1 week, and sodium propionate is gradually increased Concentration carries out conservation to 5%, by the eugonic bacterial strain finally obtained.
Further, propionic acid early period na concn is set as 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, the later period propionic acid Na concn is set as 2%, 3%, 4%, 5%, and the concentration of corresponding glucose is 0.2%, 0.3%, 0.4%, 0.5%；It is described The various salinity of minimal medium are as follows: MnSO40.0399mg/L、ZnSO4·H2O0.0428mg/L、(NH4)2MoO4·4H2O 0.0347mg/L、FeCl30.25mg/L、CaCl236.4mg/L、MgSO467.5mg/L、K2HPO4·H20108.75mg/L、 Na2HPO4·12H2O 167.4mg/L、KH2PO443.5mg/L、NH4Cl25.0mg/L。
Detailed description of the invention
Fig. 1 is Gordonia paraffinivorans ZM052 bacterial strain in LB culture medium scribing line growing state figure；
Fig. 2 is to grow for Gordonia paraffinivorans ZM052 bacterial strain 24 hours under different sodium propionate concentration conditions The absorbance value figure of bacterium solution afterwards；
Fig. 3 is GordoniaparaffinivoransZM052 strain growth situation map under the conditions of different carbon source.
Specific embodiment
The present invention is described in detail With reference to embodiment.
The instrument of screening and domestication should use after 121 DEG C of sterilizing 30min, and part vessel are gone out using 160 DEG C of hot air sterilization It is used after bacterium 120min.Operating process stringent sterilization, prevents living contaminants.Select to use minimal medium+rumen fluid+propionic acid Sodium takes the rumen fluid of 5ml in 100ml triangular flask, and the sodium propionate of 0.1-1% and the minimal medium of 40ml is added in constant temperature Shaking table 120-150rpm is protected from light culture at 30-39 DEG C, growth situation is observed after 2-5 days；Propionic acid na concn is gradually changed, It is gradually inoculated with, completes to cultivate 1 week under the conditions of the sodium propionate of various concentration as 1 round, complete the culture of 3-5 round；It will be obtained Bacterium solution cross on solid medium isolated object bacteria.
Propionic acid na concn is set as 0.25%, 0.5%, 0.75%, 1.0%, carries out the culture of 4 rounds, condition of culture For constant-temperature table 150rpm, culture is protected from light at 35 DEG C.The various salinity of minimal medium involved in the present invention are as follows: MnSO40.02-0.05mg/L、ZnSO40.02-0.05mg/L、(NH4)2MoO40.02-0.05mg/L、FeCl30.1-0.4mg/L、 CaCl220-50mg/L、MgSO450-80mg/L、K2HPO490-120mg/L、Na2HPO4100-160mg/L、KH2PO430-50mg/ L、NH4Cl15-50mg/L。
Preferably, domestication uses minimal medium+glucose+sodium propionate, takes object bacteria containing 0.1-1%'s early period Sodium propionate minimal medium is inoculated with shaking flask culture, and propionic acid na concn is gradually increased to 1%；Later period uses the sodium propionate of 1-5% Minimal medium shaking flask culture, wherein according to glucose: glucose, each acetic acid is added in the ratio that sodium propionate is 1:20-1:5 Na concn cultivation cycle is 1 week, and the concentration of sodium propionate is gradually increased to 5%, the eugonic bacterial strain finally obtained is carried out Conservation.Domestication propionic acid early period na concn is set as 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, tames later period propionic acid na concn It is set as 2%, 3%, 4%, 5%, the concentration of corresponding glucose is 0.2%, 0.3%, 0.4%, 0.5%；It is recommended to use nothing The various salinity of machine salt culture medium are as follows: MnSO40.0399mg/L、ZnSO4·H2O0.0428mg/L、(NH4)2MoO4·4H2O 0.0347mg/L、FeCl30.25mg/L、CaCl236.4mg/L、MgSO467.5mg/L、K2HPO4·H20108.75mg/L、 Na2HPO4·12H2O 167.4mg/L、KH2PO443.5mg/L、NH4Cl25.0mg/L。
Fig. 1 is Gordonia paraffinivorans ZM052 bacterial strain in LB culture medium scribing line growing state.
Fig. 2 is to grow for Gordoniaparaffinivorans ZM052 bacterial strain 24 hours under different sodium propionate concentration conditions The absorbance value of bacterium solution afterwards.Note in Fig. 2: it is followed successively by blank (sterile), headpin (glucose), No. 12 bottle (carboxymethyls from left to right Sodium cellulosate), No. 13 bottles (alkaline lignin), No. 22 bottles (naphthalene), No. 23 bottles (phenanthrene), No. 24 bottles (anthracene), No. 25 bottles (pyrene).Fig. 3 It is Gordonia paraffinivorans ZM052 strain growth situation under the conditions of different carbon source.
The present invention constructs screening and the acclimation method of tolerance propionate bacteria, proposes propionate and tolerance propionate is thin The use in conjunction of bacterium gets the tolerance propionate bacteria Gordonia paraffinivorans in one plant of cud source ZM052, it was confirmed that the bacterial strain has good growth performance in propionate, while also observing the bacterial strain in carboxymethyl cellulose Sodium, alkaline lignin, naphthalene, phenanthrene, anthracene, pyrene indicate that the bacterial strain has and are applied to raise to have growth in the culture medium of carbon source Material fermentation, the mainly aerobic fermentation of herbage and stalk, playing improves feed nutritive value (composite part carotenoid, drop Cellulose and lignin are solved, mycoprotein is provided, influences rumen microorganism) and degradation some of the contaminants (mainly polycyclic aromatic hydrocarbon Class) effect.
The above is only not to make limit in any form to the present invention to better embodiment of the invention System, any simple modification that embodiment of above is made according to the technical essence of the invention, equivalent variations and modification, Belong in the range of technical solution of the present invention.

Claims (6)

1. the tolerance propionate bacteria acclimation method in one plant of cud source, it is characterised in that: take rumen fluid, sodium propionate and nothing is added Machine salt culture medium is protected from light culture in constant-temperature table, gradually changes propionic acid na concn, is gradually inoculated with, and completes the propionic acid of various concentration It is cultivated under the conditions of sodium, bacterium solution obtained is crossed isolated object bacteria on solid medium.
2. according to the tolerance propionate bacteria acclimation method in one plant of cud source described in claim 1, it is characterised in that: take 5ml Rumen fluid in 100ml triangular flask, the sodium propionate of 0.1-1% and the minimal medium of 40ml is added in constant-temperature table 120- It is protected from light culture at 150rpm, 30-39 DEG C, growth situation is observed after 2-5 days；Propionic acid na concn is gradually changed, is gradually inoculated with, It completes to cultivate 1 week under the conditions of the sodium propionate of various concentration as 1 round, completes the culture of 3-5 round；Bacterium solution obtained is existed It crosses on solid medium isolated object bacteria.
3. according to the tolerance propionate bacteria acclimation method in one plant of cud source described in claim 1, it is characterised in that: described third Sour na concn is 0.25%, 0.5%, 0.75%, 1.0%, carries out the culture of 4 rounds, condition of culture is constant-temperature table 150rpm is protected from light culture at 35 DEG C.
4. according to the tolerance propionate bacteria acclimation method in one plant of cud source described in claim 1, it is characterised in that: the nothing The various salinity of machine salt culture medium are as follows: MnSO4 0.02-0.05mg/L、ZnSO4 0.02-0.05mg/L、(NH4)2MoO4 0.02- 0.05mg/L、FeCl3 0.1-0.4mg/L、CaCl2 20-50mg/L、MgSO4 50-80mg/L、K2HPO4 90-120mg/L、 Na2HPO4 100-160mg/L、KH2PO4 30-50mg/L、NH4Cl15-50mg/L。
5. according to the tolerance propionate bacteria acclimation method in one plant of cud source described in claim 1, it is characterised in that: early period takes Object bacteria is inoculated with shaking flask culture in the sodium propionate minimal medium containing 0.1-1%, and propionic acid na concn is gradually increased to 1%； Later period uses the sodium propionate minimal medium shaking flask culture of 1-5%, wherein according to glucose: sodium propionate is the ratio of 1:20-1:5 Glucose is added in example, and each propionic acid na concn cultivation cycle is 1 week, and the concentration of sodium propionate is gradually increased to 5%, will finally obtain Eugonic bacterial strain carry out conservation.
6. according to the tolerance propionate bacteria acclimation method in one plant of cud source described in claim 1, it is characterised in that: before described Phase propionic acid na concn is set as 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, the later period propionic acid na concn is set as 2%, 3%, 4%, 5%, the concentration of corresponding glucose is 0.2%, 0.3%, 0.4%, 0.5%；The minimal medium is various Salinity are as follows: MnSO4 0.0399mg/L、ZnSO4·H2O0.0428mg/L、(NH4)2MoO4·4H2O 0.0347mg/L、FeCl3 0.25mg/L、CaCl2 36.4mg/L、MgSO4 67.5mg/L、K2HPO4·H20 108.75mg/L、Na2HPO4·12H2O 167.4mg/L、KH2PO443.5mg/L、NH4Cl25.0mg/L。
CN201811360010.0A 2018-11-15 2018-11-15 The tolerance propionate bacteria acclimation method in one plant of cud source Pending CN109468258A (en)

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CN111778187A (en) * 2020-07-07 2020-10-16 内蒙古恒盛环保科技工程有限公司 Microbial repairing microbial inoculum and preparation method thereof

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